Simonetta Poggi

The EnVision++ system: a new immunohistochemical method for diagnostics and research. Critical comparison with the APAAP, ChemMate, CSA, LABC, and SABC techniques.

AIM: To assess a newly developed immunohistochemical detection system, the EnVision++. METHODS: A large series of differently processed normal and pathological samples and 53 relevant monoclonal antibodies were chosen. A chessboard titration assay was used to compare the results provided by the EnVision++ system with those of the APAAP, CSA, LSAB, SABC, and ChemMate methods, when applied either manually or in a TechMate 500 immunostainer. RESULTS: With the vast majority of the antibodies, EnVision++ allowed two- to fivefold higher dilutions than the APAAP, LSAB, SABC, and ChemMate techniques, the staining intensity and percentage of expected positive cells being the same. With some critical antibodies (such as the anti-CD5), it turned out to be superior in that it achieved consistently reproducible results with differently fixed or overfixed samples. Only the CSA method, which includes tyramide based enhancement, allowed the same dilutions as the EnVision++ system, and in one instance (with the anti-cyclin D1 antibody) represented the gold standard. CONCLUSIONS: The EnVision++ is an easy to use system, which avoids the possibility of disturbing endogenous biotin and lowers the cost per test by increasing the dilutions of the primary antibodies. Being a two step procedure, it reduces both the assay time and the workload.

Lymphohistiocytic T‐cell lymphoma (anaplastic large cell lymphoma CD30+/Ki‐1 + with a high content of reactive histiocytes)

We describe 13 cases of a peculiar lymphoid tumour containing very large numbers of reactive histiocytes. The tumours occurred in young patients (mean age 14.8 y) who presented with systemic symptoms and superficial lymphadenopathy. Microscopic examination revealed a diffuse effacement of lymph node structure due to the presence of histiocytes intermingled with a variable number of anaplastic large lymphoid cells. The latter, in some cases, were isolated, while in others they were arranged in clusters or were diffusely present in residual sinuses. The large anaplastic cells expressed the activation markers CD30 (Ki‐1), CD25 (interleukin‐2 receptor), CD70 (Ki‐24) and Ki‐27, as well as varying combinations of T‐associated molecules. The histiocytes expressed lysozyme and the CD11b (C3bi‐R), CD11c (p150, 95) CD14, CD68 (KPI) and Ber‐Mac3 antigens. Double staining with the antibody Ki‐67 demonstrated that the proliferating components were the CD30‐positive cells and not the histiocytes. T‐cell receptor beta gene rearrangements were shown in three cases tested. The patients responded well to aggressive chemotherapy and nine are still alive, eight in complete remission. It is suggested that the tumour represents a well‐defined clinico‐pathological entity originating from activated Tlymphocytes.

Anaplastic large cell lymphoma (CD30+/Ki-1+): results of a prospective clinico-pathological study of 69 cases

Summary. Sixty‐nine anaplastic large cell lymphomas (ALCLs) were selected from an Italian comparative trial on MACOP‐B and F‐MACHOP. As no significant difference in effectiveness of the protocols emerged, they were considered homogenously treated. The ALCLs were divided into two groups according to previously defined criteria: 41 were common type (ALCLs‐CT) and 28 Hodgkin‐related (ALCLs‐HR). T‐cell phenotype was most common (58%), while B‐cell, null and hybrid forms accounted for 27%, 13% and 2%. Clinically, ALCLs CT and HR differed as to mean age (27 v 34·3 years) and presentation; all ALCLs‐HR showed mediastinal involvement, with bulky disease in 57%, and more frequent occurrence in stage II. In contrast, ALCLs‐CT showed mediastinal masses in 58·5%, infrequently revealed bulky disease (24%), and were not specifically associated to stage. Among the ALCLs‐CT, 68·4% achieved complete remission (CR), 24·4% partial remission (PR), one (2·4%) was resistant to therapy, and two (4·8%) had fatal drug toxicity. Of the ALCLs‐HR, 67·8% reached CR, 14·3% PR, and 17·9% did not respond. In CR, ALCLs‐CT showed a greater tendency to relapse (32·1%v 14·2%). At present, 65·8% of ALCLs‐CT and 67·8% of ALCLs‐HR are alive with overall survival/disease‐free survival averages of 31/27 and 29/24 months respectively. Our data emphasize that, independently of subtype, ALCLs benefit from the application of third‐generation protocols for high‐grade non‐Hodgkins lymphomas.

Variable expression of leucocyte-common (CD45) antigen in CD30 (Ki1)-positive anaplastic large-cell lymphoma: Implications for the differential diagnosis between lymphoid and nonlymphoid malignancies

Monoclonal antibodies (mAbs) directed against the leucocyte common (CD45) antigen have been proposed as a useful tool for the differential diagnosis between malignant lymphomas (CD45+) and poorly differentiated nonhemopoietic tumors (CD45-). Thanks to the availability of mAbs directed against fixative-resistant epitopes of the CD45 molecule, this distinction can now easily be made even in routinely processed tissues. However, a small percentage of morphologically poorly defined neoplasms are difficult to diagnose even with the help of immunohistochemistry. The investigators report that 63 out of 165 anaplastic large-cell (ALC) lymphomas did not show any reactivity for the CD45 antigen in paraffin sections. In routine biopsies, the lymphomatous nature of these cases, most of which had been sent for consultation, could be always unequivocally established by demonstrating negativity for cytokeratins (mAb KL1) and clear dot-like and/or surface reactivity with the Ber-H2 mAb, which is directed against a fixative-resistant epitope of the lymphoid cell activation antigen CD30. Strikingly, 54% of the CD45-cases reacted with mAbs directed against fixative-resistant epitopes of the T cell-restricted CD45RO antigen (mAb UCHL1) or the B-restricted molecules CD45RA (mAb 4KB5) and L26 (unclustered). In order to avoid confusion of ALC lymphomas with anaplastic nonlymphoid tumors, pathologists must be aware of the existence of CD30+/CD45- ALC lymphomas, as they can mimic the above-mentioned malignancies both morphologically (due to the sinusoidal growth pattern) and phenotypically (due to the expression of EMA). The investigators conclude that the combined use of mAbs directed against fixative-resistant epitopes of the CD30, CD45RO, CD45RA, and L26 antigens and cytokeratins is essential for the correct diagnosis and treatment of these equivocal cases.

Myeloperoxidase Expression by Histiocytes in Kikuchi's and Kikuchi-Like Lymphadenopathy

Forty-five examples of Kikuchis lymphadenitis (KL), 5 Kikuchi-like lupus erythematosus lymphadenopathies, 25 nonnecrotizing lymphadenitidies (5 toxoplasmic, 5 sarcoid-like, 6 dermatopathic, 4 suppurative, 3 tubercular, 2 with sinus histiocytosis), 4 examples of hyaline-vascular Castleman disease (CD), 2 plasmacytoid monocyte tumors (PM-Ts), and 61 accessory cell neoplasms were studied by a panel of antibodies, including the PG-M1 (against a macrophage-restricted CD68 epitope) and a polyclonal anti-myeloperoxidase (MPO). In KL and Kikuchi-like lupus erythematosus lymphadenopathies, 25 to 75% of CD68(+) histiocytes co-expressed MPO. This did not occur in nonnecrotizing lymphadenitidies and accessory cell neoplasms. MPO(+)/CD68(+) elements corresponded to nonphagocytosing mononuclear cells and some crescentic macrophages and phagocytosing histiocytes. Typical PMs were MPO(-)/CD68(+) in all cases, including CD and PM-T. Our observations suggest that in KL and KL-like lymphadenopathies: 1) MPO(+)/CD68(+) blood monocytes might be attracted into tissues because of the lack or paucity of granulocytes and the need of MPO for oxidative processes; 2) PMs are more likely to be involved in the cytotoxic immune reaction than in phagocytic phenomena; 3) the peculiar phenotype of the histiocytic component can be usefully used for the differentiation from malignant lymphoma and PM-T.

Mediastinal large B‐cell lymphoma: clinical and immunohistological findings in 18 patients treated with different third‐generation regimens

We report on the immunophenotype, clinical findings and response to aggressive chemotherapy of 18 patients with mediastinal large B‐cell lymphoma (MLCL). Cases were collected from a series of 286 high‐grade non‐Hodgkins lymphomas (HG‐NHL) which, in the period September 1988 to August 1991, were enrolled in a prospective multicentre trial designed to compare the MACOP‐B and F‐MACHOP regimens. Immunostaining on frozen sections revealed a previously unrecognized phenotype, i.e. co‐expression of B‐cell (CD19, CD20, CD22, Ig‐associated dimer) and activation‐associated antigens (CD30 and CDw70) in about 60% of MLCL cases; in contrast, the activation‐associated antigens CD25 and Ki‐27 (unclustered) were consistently negative. This peculiar phenotype may reflect a derivation of the tumour from a subset of thymic activated B cells. Clinically, the patients (median age 31 years; F/M ratio 2.6) presented with bulky mediastinal mass (72%) associated with mediastinal syndrome in >50% cases; disease was stage IIA in most cases. All 18 patients received aggressive chemotherapy (F‐MACHOP 11; MACOP‐B 7). Complete response (CR) was achieved in 57.1% of cases treated with MACOP‐B. In contrast, the response of the 11 MLCL treated with F‐MACHOP was poor (CR 18.2%) as compared to that of the 135 HG‐NHL treated with the same regimen during the trial (CR 69.6%). This difference was still statistically significant after adjusting for negative prognostic factors (mediastinal mass > 10 cm plus increased LDH) and suggests that F‐MACHOP might not be the most appropriate regimen for this kind of lymphoma.

Neoplastic cells of Hodgkin's disease show differences in EBV expression between Kenya and Italy

The Epstein‐Barr Virus (EBV) has been implicated in the pathogenesis of Hodgkins disease (HD). However, the association of EBV with this disease varies greatly from series to series and from country to country. Epidemiological studies have shown differences in HD occurring in different parts of the world. In particular, it has been reported that HD in developing countries differs from HD in Western countries in terms of epidemiological, pathological and clinical characteristics. These discrepancies among populations suggest an interaction with environmental factors and a direct role of different etiological agents. At present, there are no data on the frequency of association of EBV with HD in equatorial Africa. In this study, a large series of HD cases have been collected at the University of Nairobi, Kenya, and at the Universities of Bologna and Siena, Italy. The cases have been reviewed and classified according to the REAL Classification and the presence of EBV has been assessed by in situ hybridization (ISH). A statistical difference in EBV expression was found between HD from Kenya and HD from Italy. EBV‐positive neoplastic cells were detected in 92% of Kenyan cases, whereas only 48% of Italian cases showed EBER1/2 positivity in the neoplastic cells. Our results suggest that, in Kenya, EBV plays a more direct role in the pathogenesis of HD, as it does for endemic Burkitt lymphoma.

Bone Marrow Findings Further Support the Hypothesis that Essential Mixed Cryoglobulinemia Type II is Characterized by a Monoclonal B-Cell Proliferation

One-hundred-sixteen consecutive bone-marrow biopsies were taken from 76 patients with essential mixed cryoglobulinemia type II (type II cryo), whose median follow-up was 97 months. Fifty-four out of fifty-six subjects who underwent ELISA and RIBA tests for HCV, were found to be positive. At conventional light microscopic examination, 64/76 patients showed discrete lymphoid infiltrates consisting of small elements with plasmacytoid differentiation and with frequent paratrabecular location. Thirty-nine biopsies were studied by immunohistochemistry that revealed the B-cell nature of the infiltrates (CD20+, CD45RA+, CD79 alpha+, CD3-, CD45RO-), with demonstrable monotypic Ig light-chain restriction in 22 cases. It is worthy of note that the lymphoid elements usually appeared protected against apoptosis, because of the strong expression of the bcl-2 oncogene product, and provided with a very low proliferative capacity, the Ki-67 index being lower that 3%. The latter findings are in keeping with the indolent behaviour of the clonal lymphoid population observed in type II cryo and allow some speculation as to the need for environmental stimuli for its maintenance as well as further mutagenic events for its eventual transformation into an overt lymphoma.

Anaplastic Large Cell Lymphoma: Update of Findings

The problem of anaplastic large cell lymphoma (ALCL) is extensively reviewed by depicting the clinical, pathological and biological characteristics of the four main varieties of ALCL: common, Hodgkins like/Hodgkin-related, lympho-histiocytic, and giant-cell rich. Special emphasis is given to the differential diagnosis between ALCL Hodgkin like and Hodgkins disease in the light of possible therapeutical differences.

Pyothorax-associated lymphoma: Description of the first two cases detected in Italy

BACKGROUND Pyothorax-associated lymphoma (PAL) is a rare, but distinct, clinico-pathologic entity which occurs most often in Japanese people; to the best of our knowledge, only six cases of it have been reported in Western countries. The tumour develops several decades following artificial pneumothorax or chronic pleuritis due to tuberculous infection, produces pleural effusion associated with extensive local lymphomatous infiltrates, and is sustained by a polymorphic large B-cell clonal proliferation showing EBV integration in the genoma of the neoplastic cells. PATIENTS AND METHODS Herein we describe two cases of PAL observed in Italian patients, both extensively studied on the clinical, pathological, phenotypic, virological, and molecular levels. RESULTS The two cases occurred, respectively, 45 and 50 years after therapeutic pneumothorax because of tuberculous pleuritis and were characterized by a pleural mass extending to the thoracic wall, which on histological examination were seen to consist of large elements with immunoblastic morphology. Immunohistochemistry show monotypic restriction of Ig light chains, as well as the expression of CD45, B-cell markers (CD20, CD79a, CD45RA), bcl-2 oncogene product, EBNA-2 and, partially, LMP-1. The ratio of cycling cells was extremely high as was the number of mitotic figures. In situ hybridization displayed the presence in the neoplastic cells of the EBV-related small RNAs EBER 1 and 2, which in turn, along with the positivity for EBNA-2 and LMP-1, further strengthened the close relationships between PAL and latent viral infection. Molecular studies revealed, on one hand, clonal rearrangement of the Ig heavy chain J region genes, and on the other, negativity for HHV8 in one case and positivity in the other. CONCLUSIONS These cases of PAL are the first to be documented in Italy; they serve to direct attention to the fact that this condition is not confined to Japanese people, and that its occurrence in Western countries might be underestimated.